Optimization of the Probiotic Fermentation Process of Ganoderma lucidum Juice and Its In Vitro Immune-Enhancing Potential

Fermented products have recently garnered substantial interest in both research and commercial contexts. Although probiotic fermentation is predominantly practiced with dairy, fruits, vegetables, and grains, its application to dual-purpose food-medicine materials like Ganoderma lucidum has been comparatively underexplored. In this study, Ganoderma lucidum fermented juice (GFJ) served as the substrate and was fermented with five probiotic strains. The optimal inoculation ratios—determined by employing a uniform design experiment—were as follows: Bifidobacterium animalis 6.05%, Lacticaseibacillus paracasei 9.52%, Lacticaseibacillus rhamnosus 6.63%, Pediococcus pentosaceus 21.38%, and Pediococcus acidilactici 56.42%. Optimal fermentation parameters established by response surface methodology included 24 h of fermentation at 37 °C, a final cell density of 5 × 106 CFU/mL, and a sugar content of 4.5 °Brix. Experiments with RAW264.7 macrophages revealed that GFJ significantly promoted both phagocytic activity and nitric oxide (NO) secretion, indicating enhanced immune characteristics as a result of fermentation. Untargeted metabolomics profiling of GFJ across different fermentation stages showed upregulation of functional metabolites, including polyphenols, prebiotics, functional oligosaccharides, and Ganoderma triterpenoids (GTs)—notably myricetin-3-O-rhamnoside, luteolin-7-O-glucuronide, raffinose, sesamose, and Ganoderma acids. These increments in metabolic compounds strongly correlate with improved functional properties in GFJ, specifically heightened superoxide dismutase activity and immunomodulatory capacity. These results highlight an effective approach for developing functionally enriched fermented products from medicinal fungi, with promising applications in functional food and nutraceutical industries.